Monitoring biomolecular and biochemistry process in organisms is a fundamental issue in biosensing, which has applications from fundamental biological research to clinical diagnostics. Mitochondria, a membrane-bound organelle found in most eukaryotic cells, play important roles in numerous vital cellular processes, such as energy supplying, reactive oxygen species generation, signaling, cellular differentiation and cell death. The mitochondrial network displays remarkable plasticity during development of certain tissues. The morphology of mitochondria is affected by cell type, cell-cycle stage, and intracellular metabolic state, which in turn contributes to cell functioning. Recent reports show that the mitochondria are also crucially involving in various pathologies, from Alzheimer's disease to cancer. Thus, development of a convenient and efficient mitochondrial imaging method is of great fundamental importance for understanding cell biochemistry process and early diagnosing disease.
Fluorescence techniques are particularly well suited for biological application, due to their non-invasive feature and high sensitivity. So far, a few fluorescent dyes have been developed for mitochondrial imaging, such as rhodamines, rosamines, carbocyanines, and BODIPY dyes, with some showing two-photon emission (TPE) properties. However, most mitochondrial probes, such as BODIPY dyes, give strong fluorescence signals in buffer solution. During the application, the unbound probes must be washed off to eliminate the strong residual signal from the free dyes to improve the signal-to-noise (S/N) ratio. The time-consuming washing process will inevitably delay the acquisition of microscopic data. In addition, the required post-application washing procedure could alter the cell environment and hamper the probe's ability to monitor mitochondrial changes in real-time. This is because the number and subcellular locations of mitochondria will dramatically change with the cell metabolic demands. Moreover, since mitochondria are directly associated with cytoactivity, the dying cells could be removed during the washing process, adversely affecting the monitor of whole cell apoptosis cycle. In order to overcome the deficiency, aggregation-induced emission (AIE) dyes have been used to minimize the fluorescence signals of free dyes. The operation of AIE dyes, however, requires the significant accumulation of dye molecules on the cells to form aggregation. It remains a challenge to develop a novel strategy that permits the specific mitochondria labeling without the post application washing, thereby enabling continuous observation of the entire biochemistry process without interruption. Although an AIE dye has been used to track the mitochondria without washing process, it is desirable to identify a new mechanism that does not require the accumulation of dye molecules on the cells, which usually takes a longer time (e.g. 20 min) and high concentration to stain a target.
Lysosomes, another component in eukaryotic cells, are important part of the endocytic system of the cell and serve a degradative function. Lysosomes are membrane-bound organelles with an acidic interior (pH of about 4.5), and contain approximately 50 different degradative enzymes that degrade the material that is brought into the cell. The degradation process is not random and in a number of instances is targeted. Deficiency in a single lysosomal enzyme could prevent breakdown of target molecules, which consequently accumulate within the lysosomes and often give rise to clinical symptoms. Deficiencies in lysosomal enzymes cause abnormal storage of macromolecular substrates, which are known as lysosomal storage diseases.
Most lysosomal sensors are weak bases (by including an amino group) that can be used to accumulate dyes in acidic organelles. Upon entering the acidic organelles, the protonation also reduces (or removes) the photoinduced electron transfer (PET) from the attached amino groups, thereby increasing the fluorescence intensity of the fluorescent dyes. For example, the commercial LysoTracker®, such as Red DND-99, has an amino group to concentrate the sensor to lysosome organelles. Unfortunately, the existing lysosomal probes often exhibit high background fluorescence signals, due to incomplete fluorescence quenching. The conditions that require the PET to switch off completely at the narrow pH (about 4.5) also raise a challenge.
Thus, a need remains in the art for improved biological imaging agents for the imaging organelles that do not suffer from one or more of the above mentioned deficiencies.